Project Summary One-fifth of the global population is exposed to some type of major circadian disruption, including shift work, frequent international travel, or disrupted sleep patterns. Disruption of circadian rhythms can increase the risk of cardiovascular disease, diabetes, and infertility. In particular, female fertility is very susceptible to circadian perturbations because ovulation is regulated in part by the suprachiasmatic nucleus (SCN), the master timekeeper of the brain. The hypothalamic preovulatory luteinizing hormone (LH) surge is a distinct rhythmic process that depends on estrogen and circadian input. Estrogen primes the kisspeptin (Kiss1) neurons in the anteroventral periventricular nucleus to secrete kisspeptin, which potently activates gonadotropin-releasing hormone (GnRH) neurons to release a bolus of GnRH, eliciting a surge of LH to prompt ovulation. Interestingly, in rodents and humans, the LH surge is restricted to the end of the subjective night. This phenomenon is believed to be due to a direct daily signal from the SCN that initiates the preovulatory surge when estrogen is sufficient. Two SCN projections have been described: One from vasoactive inhibitory peptide (VIP) neurons to GnRH neurons, and another from arginine vasopressin (AVP) neurons to Kiss1 neurons in the anteroventral periventricular nucleus. While the peptides VIP and AVP have both been shown to modulate the timing of the LH surge in vivo, the function of the SCN-GnRH and SCN-Kiss1 projections as a whole has never been studied. The overall goal of this project is to characterize the SCN-GnRH and SCN-Kiss1 projections in vivo to further our understanding of how circadian rhythms affect fertility. Aim 1 disrupts the circadian rhythms in VIP and AVP SCN neurons. Circadian manipulation of both populations independently will determine how disrupted clock function may affect fertility, the LH surge, and temporal activation of GnRH and Kiss1 neurons. Aim 2 uses optogenetics to examine the effect of direct stimulation of the SCN-GnRH and SCN-Kiss1 projections on LH release and activation of GnRH and Kiss1 neurons, which will demonstrate the sufficiency of either projection to drive LH release in vivo. Aim 3 uses a Gi-coupled designer receptor exclusively activated by designer drugs (DREADD) to transiently inhibit either VIP or AVP SCN neurons prior to the LH surge, which will establish the necessity of the SCN-GnRH or SCN-Kiss1 projection, respectively. All of these aims will provide novel and fundamental insight into the temporal regulation of the LH surge by the SCN in vivo. The proposed research will not only establish the roles of the SCN-GnRH and SCN-Kiss1 projections on the LH surge, but will also provide a basis to further explore the effects of disrupted circadian rhythms on reproductive function. The experiments proposed will broaden our understanding of the temporal mechanisms gating the LH surge, and thus fertility, and may direct treatments for infertility, anovulation, and idiopathic hypogonadotropic hypogonadism.